RNA-Protein Interaction Detection and Applications in Living Cells

捕獲活細胞中RNA和蛋白相互作用方法及應用

Student thesis: Doctoral Thesis

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Award date2 Jan 2020

Abstract

RNA biology is coordinated by the dynamic interactions of RNAs and RNA-binding proteins. RNAs function together with specific binding proteins to determine post-transcriptional processes including RNA splicing, cleavage and polyadenylation, RNA modification, trafficking, translation, and stability. The interactions of RNA-protein are pervasive in cells. However, detection of RNA-protein interactions with existing methods frequently meet various obstacles or inefficiency. Here, we describe RNA-bound protein proximity labeling (R-PL), which uses a proximity-dependent protein labeling method to detect RNA-protein interaction. In this method, we chose the well-studied RNA binding protein PUF to test for current proximity labeling enzymes birA* and APEX2. A new version of birA*, BASU was employed to validate that PUF protein binds its RNA motif. We further optimized the R-PL labeling system by using inducible expression system. The R-PL(λN-BASU) labeling experiments display higher signal-to-noise ratios. Later, we tested for R-PL (λN-APEX2) in the detection of RNA-protein interaction. By comparison, proximity labeling assays suggest that R-PL (λN-BASU) is more suitable than R-PL (λN-APEX2) in the detection of RNA-protein interaction in live cells. Interestingly, the results also reveal that proximity labeling is probably capable of biotinylating proximate nascent peptide for the first time.

In the second part, we present a study of protein-based siRNA delivery. siRNAs hold great promise for gene-targeted therapeutic but one major obstacle is the delivery to disease sites and into the cell cytosol. The unique complex of p19 binds to ~21nt dsRNA motivated us to employ RNA-protein interaction complex as vehicle for siRNA delivery. p19 protein can be directed to desired place in cell by fusing with various signal peptides. Our proof-of-concept experiment showed that the complex of p19 protein and its bearing siRNAs, expressed and purified from bacteria, could mediate reporter gene knockdown in mammalian cells. Further studies have been carried out to optimize p19 protein-based siRNA delivery for gene silencing efficiency: (1) p19 is fused with cell-penetrating peptide (CPP) and CPP drives p19-siRNA complex to penetrate cells; (2) destabilization peptide promotes degradation of fusion protein after entry into cell and releases bound siRNAs in mammalian cell. In the future, we will further employ bacterial strains that carry p19 protein bearing siRNAs directly into tumor cells in animal model.